Summary
Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.
Abstract
Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.
Introduction
Epithelial and endothelial cells function as cellular boundaries, separating the apical and basolateral sides of the body. If they are connected through tight junctions, passive substance diffusion through the paracellular spaces is restricted1, resulting in the formation of a selectively permeable barrier. Several artificial barrier systems have been developed2 using microvascular endothelial cells (HBMEC, blood-brain barrier3,4,5,6,7), choroid plexus epithelial cells (HIBCPP/PCPEC, blood-cerebrospinal fluid barrier8,9,10,11,12,13,14), colorectal adenocarcinoma cells (Caco-2, gastrointestinal models15), or airway/alveolar cell lines (pulmonary models16,17). These systems typically consist of cells grown in a monolayer on permeable membranes (i.e., filter insert systems) to allow access to the apical and basolateral sides. It is important that the integrity of the model system matches the in vivo conditions. Hence, several techniques have been developed to analyze barrier function by measuring paracellular diffusion of tracer compounds across the cell layer. These substances include radiolabeled sucrose, dye-labeled albumin, FITC-labeled inulin, or dye-labeled dextrans2. However, chemical dyes can make cells unusable for further experiments. To monitor barrier systems noninvasively, measurement of transepithelial/transendothelial electrical resistance (TEER) across a cellular monolayer can be used2,18,19. Because bipolar electrode systems are influenced by the electrode polarization impedance at the electrode-electrolyte interface, tetrapolar measurements are generally used to overcome this limitation20. The underlaying technique is a four-terminal sensing (4T) that was first described in 1861 by William Thomson (Lord Kelvin)21. In brief, the current is injected by a pair of current-carrying electrodes while a second pair of voltage-sensing electrodes is used to measure the voltage drop20. Nowadays, so-called chopstick electrodes consist of a pair of double electrodes, each containing a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current2. The electrical impedance is measured between the apical and the basolateral compartment with the cell layer in between (Figure 1). A square wave signal at a frequency of typically 12.5 Hz is applied at the outer electrodes and the resulting alternating current (AC) measured. Additionally, the potential drop across the cell layer is measured by the second (inner) electrode pair. Electrical impedance is then calculated according to Ohm's law. TEER values are normalized by multiplying impedance and cell layer surface area and are typically expressed as Ω ∙ cm2.
There are systems in which cells and electrodes are arranged in a more sophisticated way, but are also based on the 4T measuring principle and can be used with the same measurement devices. EndOhm systems, for example, in which the filter is inserted, contain a chamber and cap with a pair of concentric electrodes with the same structure as the chopstick electrode. The shape of the electrodes allows for a more uniform current density flow across the membrane, thereby reducing variation between readings. Even more complex (but also more accurate) is an Ussing chamber, where a cell layer separates two chambers filled with Ringer's solution22. The chamber itself can be gassed with oxygen, CO2, or N2, and stirred or supplemented with experimental substances. As ion transport across the cell layer occurs, a potential difference can be measured by two voltage-sensing electrodes near the tissue. This voltage is cancelled out by two current-carrying electrodes placed next to the cell layer. The measured current will then give the net ion transport and the transepithelial resistance, which reflects barrier integrity, can be determined22. TEER measurement can also be applied on body-on-a-chip systems that represent barrier-tissue models23,24. These systems mimic in vivo conditions of the cells and often consist of several types of cells, stacked on top of each other in layers.
The following protocol explains how to set up a cost-effective and reliable voltammeter with programmable output frequency that produces no statistically significant differences in TEER compared to commercially available measurement systems.
Subscription Required. Please recommend JoVE to your librarian.
Protocol
1. Assembly of a basic volt-amperemeter for TEER measurement
- Prepare a standard USB charger as the 5 V D.C. power supply, a USB extension cord, a microcontroller that will be used as a programmable square wave generator, two standard multimeters that are able to measure alternating current and voltage as root mean square (True-RMS), four cables with banana plugs, a telephone extension cord with a RJ14 female connector including six pins with the inner four wired (6P4C), two short cables, a luster terminal, a 120 kΩ pre-resistor, wire end ferrules, and soldering lugs. The tools required are an insulation stripper, a crimping tool, and a soldering iron.
- First, connect the USB extension to the microcontroller board.
- Strip the end insulation of two short cables. Solder one side per cable either directly to pins 0 and 2 of the microcontroller or to soldering lugs, which in turn are clipped on the respective pins. Crimp the other ends to the wire end ferrules and connect them to a luster terminal as depicted in Figure 1.
- Link the banana plugs to the multimeters. Strip and crimp the other end of each of the four cables.
- Cut the telephone extension cord in two pieces and dismantle and crimp the conductors of the side containing the female connector. Check for the continuity of the conductors and pins.
- The first multimeter will be used to measure current in µA (note that the AC mode has to be set explicitly). Connect it in a series with a 120 kΩ pre-resistor to pins five and six of the RJ14 connector, corresponding to the outer electrode pair of the chopstick electrode.
- Finally, link the second multimeter, which will be used to measure the transepithelial voltage drop in mV, via the luster terminal to pins three and four of the RJ14 connector, corresponding to the inner electrode pair of the chopstick electrode.
- If desired, mount the installation in a chassis.
2. Programming the microcontroller
- Modify the provided source code (supplemental coding file 1) as needed. In the given form, pins 0 and 2 will alternate between ground and +5 V with 40 ms half-time of oscillation. Thus, a square wave signal with an amplitude of 5 V and a frequency of approximately 12.5 Hz will be generated. The real values may differ due to the inaccuracy of the microcontroller's time emitter.
- Connect the microcontroller to a desktop computer via a USB port and upload the source code with matching software25.
3. Recording of voltage oscillograms (optional)
- Bypass pins five and six of the RJ14 connector with a 1 kΩ test resistor and connect to an oscilloscope.
- Check for the frequency, peak voltage, and waveform. Digitize and export the data.
- If desired, record oscillograms from a reference device (EVOM) and the self-assembled voltammeter for comparison.
NOTE: In this case, the data was recorded with a Digital Storage Scope HM 208. Being a very basic digital oscilloscope, the image could be internally digitized (frozen) but had to be plotted using an analogue PM 8143 X-Y recorder. The image was subsequently scanned.
4. Cell cultivation and TEER measurement
- Seed Human Choroid Plexus Papilloma (HIBCPP) cells on cell culture filter inserts with a pore size of 3 µm in DMEM/F12 (see Table of Materials) containing 10% fetal calf serum9. Grow the cells at 37 °C in a water saturated atmosphere containing 5% CO2 as described by Dinner et al.9.
- When the filters reach an impedance of 70 Ω ∙ cm2, change to serum-free DMEM/F12 and define the timepoint as Day 0.
- Connect the electrode to the RJ14 port of the self-assembled voltammeter and plug in the USB power supply. Set the multimeters to AC voltage mode (mV) and AC current mode (µA), respectively.
- Alternatively, connect the electrode to a commercially available reference device and turn on according to the manufacturer's instructions.
- Sterilize the electrode in 80% ethanol for 10 min and equilibrate in the appropriate medium for another 10 min.
- Put the electrode in both compartments of a cell culture filter insert system (the longer part of the electrode in the lower compartment and the shorter part in the upper compartment) containing a HIBCPP cell layer until the measurement values remain constant.
- For a reference device, note the impedance directly or calculate the impedance according to Ohm's law (R = U/I) for the self-assembled voltammeter. Be aware that electrode angle affects the measurements.
- Repeat the TEER measurement (steps 3−6) from Day 0 until Day 4.
Subscription Required. Please recommend JoVE to your librarian.
Representative Results
To compare the operation of a self-assembled voltammeter with its commercially available counterpart, a voltage oscillogram of both devices was recorded.
As shown in Figure 2A, the reference instrument generated a square wave signal with an amplitude of 80 mV and an oscillation time of 80 ms, which corresponds to a frequency of 12.5 Hz, when operating on-load with a 1 kΩ test resistor.
In contrast, the microcontroller of the self-assembled device switched the supply voltage to a square wave signal with an amplitude of 5 V (Figure 2B) if no pre-resistor was set in. It became apparent that the resulting current destroys any barrier function and is not applicable for cell culture experiments (data not shown). A further issue is that, in this setup a 1 kΩ test resistor caused an overload with a resulting decline of voltage (Figure 2B). Additionally, the effective oscillation time of the microcontroller was 60 ms (frequency = 16.7 Hz) and thereby differed from the programmed delay time due to the inaccuracy of the time emitter. If a 120 kΩ preresistor was installed, the amplitude decreased to a value of 40 mV, which was suitable for cell culture (Figure 2C). As seen in the oscillogram, signal-to-noise ratio was considerably impaired (Figure 2C) but did not affect measurements noticeably.
Both devices were used to determine the impedance of an artificial blood-cerebrospinal fluid barrier (simplified circuit diagram shown in Figure 2D). HIBCPP cells were cultivated on cell culture filter inserts and TEER was measured over 6 days: starting one day before cells were moved to serum-free conditions (Day -1) and up to 4 days after changing the medium (Day 4). All measurements were done in quadruplicates using four HIBCPP filters prepared in the same manner. Similar values were obtained for the reference instrument and the self-assembled voltammeter (Figure 3). Measurements were reproducible, and standard deviations were within the same range. TEER values ranged from 20−550 Ω ∙ cm2. Using 0.33 cm2 filters, this equates to an absolute impedance of 83−1,660 Ω.
Figure 1: Layout diagram of a basic volt-amperemeter for TEER measurement. Please click here to view a larger version of this figure.
Figure 2: Oscillograms and measurement setup. (A) Commercially available EVOM. (B) Self-assembled voltammeter without pre-resistor. (C) Self-assembled voltammeter with 120 kΩ pre-resistor. (D) Circuit diagram of measurement setup. Note that Celectrode only appears in the electrical circuits when bipolar systems are used. Please click here to view a larger version of this figure.
Figure 3: TEER measurements of HIBCPP cell layers on cell culture filter inserts before switching to serum-free culture medium (Day -1), on the day of switching (Day 0), and up to 4 days after (Days 1−4). Error bars indicate the standard deviation of the four HIBCPP filters that were prepared in the same manner. Please click here to view a larger version of this figure.
Supplemental Coding File 1. Please click here to download this file.
Supplemental Coding File 2. Please click here to download this file.
Supplemental Coding File 3. Please click here to download this file.
Subscription Required. Please recommend JoVE to your librarian.
Discussion
Before a self-made voltammeter can be used in a daily routine, it is essential to check the device for proper function. In our case, a half-time of oscillation of 40 ms (12.5 Hz) was programmed, but the effective oscillation time turned out to be 60 ms (16.7 Hz). This inaccuracy of the microcontroller's time emitter had no detectable impact on TEER measurements. It might be best to determine the actual frequency using the frequency setting of one of the multimeters. If any deviation is found, the source code can be adjusted accordingly. Further, it is strongly recommended to check whether a test resistor or other defined setups give correct and reproducible results. If working with artificial cellular barrier systems, it might be best to always correlate molecule flux with impedance measurement.
In this case, the applied current was limited using a 120 kΩ pre-resistor. Assuming that typical TEER values range from 100 Ω−2,000 Ω, the voltage drop across the cell layer can be calculated to be 4−83 mV. A TEER of 1 kΩ was simulated by a test resistor and the resulting potential drop was confirmed to be 40 mV (Figure 2C).
Commercially available devices often provide a measurement range switch to toggle the pre-resistor and thus limit the output current to different values. In this case, it is feasible to install different pre-resistors or to even replace the resistor with a potentiometer.
The shown setup represents a cost-effective alternative to commercially available instruments for TEER measurement. Values that have been measured with the self-assembled voltammeter were comparable to the reference device over a broad range. The same is true for the standard deviations. The noise in the square wave signal did not affect measurements notably. The protocol can support scientists who are restricted by limited financial resources or who want to perform preliminary experiments at low costs.
Further, the microcontroller can be easily programmed to different output frequencies. This may be beneficial, as the apparent impedance consists of Rmedium, RTEER, as well as the capacity Ccell layer26 (Figure 2D). Additionally, Celectrode appears if bipolar systems are used, whereas the influence from the electrode polarization impedance is reduced in tetrapolar systems. This means that the measured impedance will be dominated by RTEER at low frequencies and, in bipolar systems, by the capacity of the electrodes, whereas at high frequencies the total impedance converges to the resistance of the medium26,27. In between, the impedance is influenced by Ccell layer, which is therefore accessible using electrical impedance spectroscopy28.
We provide two (untested) example codes to give an idea how the device could be optimized or reprogrammed for different applications. First, a very basic impedance spectroscopy could be realized by alternating the output frequency in 20 second intervals between 12.5, 500 and 5000 Hz (supplemental coding file 2). In this case, a tetrapolar20,28 or bipolar27 electrode could be used. Applied frequency could be shown by the build-in multimeter (or any display or LED connected to the microcontroller). Second, the device could be used to measure conductivity of buffers and media. This is typically done using tetrapolar electrodes with high frequencies in a range of 1-110kHz. The code in supplemental coding file 3 contains no delay time and (with our device) generated a frequency of approximately 70kHz.
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The authors have no competing financial interests or other conflicts of interest.
Acknowledgments
The authors would like to thank Herman Liggesmeyer and Marvin Bende for their expert advice in electrotechnics and informatics.
Materials
| Name | Company | Catalog Number | Comments |
| 120 kOhm resistor | General (generic) equipment | ||
| Banana plug cables | General (generic) equipment | ||
| Cables | General (generic) equipment | ||
| Chopstick electrode | Merck Millicell | MERSSTX01 | |
| Chopstick electrode (alternative) | WPI World Precision Instruments | STX2 | |
| Crimping tool | General tool | ||
| Digispark / ATtiny85 | AZ-Delivery Vertriebs GmbH | Digispark Rev.3 Kickstarter | |
| DMEM:F12 | Gibco (Thermo Fisher) | 31330038 | |
| Fetal calf serum (FCS)/Fetal Bovine Serum (FBS) | Life Technologies | 10270106 | |
| Filter inserts 3µm translucent | Greiner Bioone | 662631 | |
| HIBCPP | Hiroshi Ishikawa / Horst Schroten | ||
| Insulation stripper | General tool | ||
| Luster terminal | General (generic) equipment | ||
| Oscilloscope | HAMEG | Digital Storage Scope HM 208 | |
| Plotter | PHILIPS | PM 8143 X-Y recorder | |
| Software Arduino | https://www.arduino.cc | Arduino 1.8.9 | |
| Soldering iron | General tool | ||
| Soldering lugs | General (generic) equipment | ||
| Telephone cable with RJ14 (6P4C) connector | General (generic) equipment | ||
| Test resistor | Merck Millicell | MERSSTX04 | |
| True-RMS multimeters | VOLTCRAFT | VC185 | |
| USB charger | General (generic) equipment | ||
| USB extension cord | General (generic) equipment | ||
| Voltohmmeter for TEER measurement | WPI World Precision Instruments | EVOM | |
| Voltohmmeter for TEER measurement (alternative) | Merck Millicell | ERS | |
| Wire end ferrules | General (generic) equipment |
References
- Matter, K., Balda, M. S. Functional analysis of tight junctions. Methods. 30, 228-234 (2003).
- Srinivasan, B., et al. TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation. 20, 107-126 (2015).
- Daniels, B. P., et al. Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier. Journal of Neuroscience Methods. 212, 173-179 (2013).
- Weksler, B. B., et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. Federation of American Societies for Experimental Biology Journal. 19, 1872-1874 (2005).
- Lippmann, E. S., Al-Ahmad, A., Azarin, S. M., Palecek, S. P., Shusta, E. V. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Scientific Reports. 4, 4160 (2014).
- Stins, M. F., Badger, J., Sik Kim, K. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microbial Pathogenesis. 30, 19-28 (2001).
- Muruganandam, A., Herx, L. M., Monette, R., Durkin, J. P., Stanimirovic, D. B. Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier. Federation of American Societies for Experimental Biology Journal. 11, 1187-1197 (1997).
- Ishiwata, I., et al. Establishment and characterization of a human malignant choroids plexus papilloma cell line (HIBCPP). Human Cell. 18, 67-72 (2005).
- Dinner, S., et al. A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side. Journal of Visualized Experiments. , (2016).
- Schwerk, C., et al. Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier. PLoS One. 7, e30069 (2012).
- Tenenbaum, T., et al. Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro. Cellular Microbiology. 11, 323-336 (2009).
- Gath, U., Hakvoort, A., Wegener, J., Decker, S., Galla, H. J. Porcine choroid plexus cells in culture: expression of polarized phenotype, maintenance of barrier properties and apical secretion of CSF-components. European Journal of Cell Biology. 74, 68-78 (1997).
- Haselbach, M., Wegener, J., Decker, S., Engelbertz, C., Galla, H. J. Porcine Choroid plexus epithelial cells in culture: regulation of barrier properties and transport processes. Microscopy Research and Technique. 52, 137-152 (2001).
- Strazielle, N., Ghersi-Egea, J. F. Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules. Molecular Pharmaceutics. 10, 1473-1491 (2013).
- Hilgendorf, C., et al. Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport. Journal of Pharmaceutical Sciences. 89, 63-75 (2000).
- Mathia, N. R., et al. Permeability characteristics of calu-3 human bronchial epithelial cells: in vitro-in vivo correlation to predict lung absorption in rats. Journal of Drug Targeting. 10, 31-40 (2002).
- Fuchs, S., et al. Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C. Cell and Tissue Research. 311, 31-45 (2003).
- Furie, M. B., Cramer, E. B., Naprstek, B. L., Silverstein, S. C. Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current. The Journal of Cell Biology. 98, 1033-1041 (1984).
- Hidalgo, I. J., Raub, T. J., Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology. 96, 736-749 (1989).
- Yeste, J., et al. Geometric correction factor for transepithelial electrical resistance measurements in Transwell and microfluidic cell cultures. Journal of Physics D Applied Physics. 49 (37), 3754 (2016).
- Northrup, E. VI: The Measurement of Low Resistance. Methods of Measuring Electrical Resistance. , McGraw-Hill. 100-131 (1912).
- Li, H., Sheppard, D. N., Hug, M. J. Transepithelial electrical measurements with the Ussing chamber. Journal of Cystic Fibrosis. 3 (Suppl 2), 123-126 (2004).
- Griep, L. M., et al. BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function. Biomedical Microdevices. 15, 145-150 (2013).
- Esch, M. B., et al. On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices. Biomedical Microdevices. 14, 895-906 (2012).
- Arduino IDE. Arduino Web Editor. , https://www.arduino.cc/en/Main/Software (2019).
- Benson, K., Cramer, S., Galla, H. J. Impedance-based cell monitoring: barrier properties and beyond. Fluids and Barriers of the CNS. 10, 5 (2013).
- Hufnagl, M. Time Resolved Transepithelial Impedance Spectroscopy Of Caco 2 Monolayers Relying on Lithographically Patterned Basolateral Electrode Cell Arrays. , University of Vienna. (2010).
- Guimerà, A., Gabriel, G., Parramon, D., Calderón, E., Villa, R. Portable 4 Wire Bioimpedance Meter with Bluetooth Link. World Congress on Medical Physics and Biomedical Engineering. International Federation of Medical and Biological Engineering Proceedings. Dössel, O., Schlegel, W. C. 25/7, Springer. Berlin, Heidelberg. (2009).
